Chronic myeloid leukemia (CML) is a hematopoietic malignancy characterized by a (9;22) translocation, which generates the constitutively active BCR-ABL tyrosine kinase. BCR-ABL is the critical mediator of disease pathogenesis, exhibiting constitutive kinase activity that drives survival and proliferation through multiple downstream pathways. First-line therapy with the ABL kinase inhibitor imatinib leads to durable responses in the majority of patients with CML, although a substantial number of patients develop resistance to therapy. The second-generation ABL inhibitors nilotinib and dasatinib have proven largely effective as salvage therapies in this clinical scenario. However, the gatekeeper BCR-ABLT315I mutant is insensitive to all three approved drugs, and new ABLT315I inhibitors are in development. Recent clinical reports suggest that sequential ABL tyrosine kinase inhibitor therapy may select for BCR-ABLT315I-inclusive compound mutants (two mutations within the same BCR-ABL molecule) that confer high-level resistance. A cell-based screen will be used to identify BCR-ABL compound mutations that confer resistance to ABLT315I inhibitors, and strategies to minimize emergence of compound mutants will be explored. A second subset of patients exhibits disease progression via a BCR-ABL-independent, oncogene switching mechanism, and the molecular events leading to this phenomenon remain unidentified. To better understand this process, the combination of functional screens using panels of siRNA and kinase inhibitors with high-throughput sequencing and microarray analysis will accelerate the identification of molecular mechanisms of BCR-ABL-independent resistance. To gain a more complete understanding of how to maximize efficacy of ABL kinase-inhibitor-based therapy, it is important to determine the molecular mechanism and kinetics by which these inhibitors commit CML cells to undergo apoptosis. The surprising clinical observation that dasatinib is effective in vivo despite only transient inhibition of BCR-ABL kinase activity challenges the dogma that kinase inhibitor efficacy is predicated on continual target inhibition. A detailed investigation of the core requirements for an ABL kinase inhibitor to irrevocably commit BCR-ABL-positive cells to apoptosis in CML will shed light on this process. Finally, further insight into the regulation of ABL kinase activity in the context of the multimeric oncogenic kinase BCR-ABL will be critical to improvements in our understanding of the biology and treatment of CML. We will apply a systematic approach to generate, screen, and pursue BCR-ABL protein constructs that are candidates for x-ray crystallography. Structural studies would enhance our understanding of the structural basis for activation and regulation of this oncogenic kinase. Thus, this proposal centers on: 1) elucidation of resistance mechanisms in CML, 2) identification of critical apoptosis commitment pathways associated with tyrosine kinase inhibitor sensitivity, and 3) improving our understanding of the regulation of kinase activity of BCR-ABL. This knowledge will inform and improve the disease management for patients with CML and other malignancies.